A CHEMILUMINESCENT MICROPLATE ASSAY TO DETECT DNA-DAMAGE INDUCED BY GENOTOXIC TREATMENTS

A damaged DNA detection assay (3D assay) using plasmid DNA adsorbed on sensitized microplates as the substrate for an in vitro repair reacti on is presented. DNA lesions are repaired by the excision repair pathw ay which implies an incision-excision reaction followed by DNA repair synthesis. In the 3D assay, ate took advantage of (i) plasmid DNA adso rption on polylysine-coated microplates that allowed various DNA-damag ing treatments; (ii) a protein extract that reproduced the repair reac tion in vitro; (iii) incorporation of digoxigenylated deoxynucleotide monophosphate during the DNA polymerization step which was quantified by a chemiluminescent reaction. Under experimental conditions for quan titative DNA adsorption, a dose-response relationship between the exte nt of DNA modification and the repair synthesis activity was found. Op timization of the biochemical parameters with UVC light-induced DNA le sions allowed the detection of about one photoproduct per plasmid circ le. This new assay that permits a quick and easy assessment of DNA dam age is applicable to the screening of genotoxic compounds and to the t esting of DNA-damaging treatments. (C) 1995 Academic Press, Inc.